N-linked glycosylation of NS1 protein modulates progeny virion assembly in orthoflaviviruses

A1 PLOS Pathogens · 2026-07-09

by Senzhao Zhang, Xuedan Tang, Zhen Wu, Hantai Tan, Yan Zhang, Wangyang Tan, Yu He, Tao Wang, Mingshu Wang, Renyong Jia, Dekang Zhu, Mafeng Liu, Xinxin Zhao, Qiao Yang, Ying Wu, Shaqiu Zhang, Juan Huang, Xumin Ou, Di Sun, Bin Tian, Anchun Cheng, Shun Chen Tembusu virus (TMUV) is a mosquito-borne avian virus belonging to the genus Orthoflavivirus within the family Flaviviridae . The nonstructural protein 1 (NS1) of TMUV is a secretory protein containing three N-linked glycosylation sites at residues N130, N175, and N207. Using a reverse genetics system and site-directed mutagenesis, we revealed that NS1 deglycosylation impairs the proliferation of recombinant TMUV (rTMUV) in multiple cell types. By performing subcellular fractionation assay, we observed that NS1 deglycosylation significantly impairs viral assembly. Besides, NS1 deglycosylation impairs the thermostability of NS1 dimers but not their formation. Tunicamycin treatment and enzyme-linked immunosorbent assays demonstrated that deglycosylated NS1 significantly induces the endoplasmic reticulum (ER) stress, which in turn reduces the secretion of NS1. Immunofluorescence and coimmunoprecipitation assays further demonstrated that deglycosylated NS1 is largely retained in the ER and enhances its interaction with the E protein. Retention using selective hooks (RUSH)-based live-cell imaging assay revealed that NS1 deglycosylation disrupts the trafficking of E protein from the ER to the Golgi apparatus. In addition, cycloheximide chase analysis showed that NS1 deglycosylation impairs its solubilization and then causes rapid degradation of NS1 and E protein by the host proteasomal pathway. Notably, the efficient viral assembly through NS1 glycosylation is a common feature among flaviviruses (West Nile virus and Yellow Fever virus). Our results consistently demonstrated that the glycosylation modification process of NS1 is highly synchronized with the maturation and assembly of viral particles during their trafficking from the ER to the Golgi apparatus. Collectively, our study confirms that NS1 glycosylation of orthoflavivirus species regulates progeny virion assembly by modulating the NS1-E interaction.

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